1. Field of the Invention
The present invention relates to an improved process for regenerating soybeans (Glycine max (L.) Merrill). More particularly, the invention relates to the use of tissue and cell culture for the regeneration of soybean plantlets for many soybean varieties. The invention also relates to the media used in this process.
2. Description of the Prior Art
Plant regeneration from cells in culture is essential for the application of somatic hybridization, for the production of new varieties through somoclonal variation and for the use of genetic engineering in producing new varieties. Although plants can be regenerated from single cells of a large number of crop species, the efforts with soybean have generally been unsuccessful.
In recent years, plant cell culture successes have had a considerable influence on the understanding of the respective roles of cell and organism in control of plant growth and development. Isolated plant cells have been shown to be amenable to in vitro culture and complete plants could be regenerated from cultures derived from somatic cells, either directly via somatic embryogenesis or indirectly via organogenesis. Generally the regeneration pathway of choice is determined empirically by the manipulation of extrinsic factors, especially growth regulators. Early investigations of certain plant species have suggested that exogenous auxin concentration is the major factor controlling somatic embryogenesis, such that its reduction leads to the initiation of embryoid formation. In other species, exposure to a definite balance of auxin and cytokinin leads to the formation of organogenesis (shoots, then roots).
Phillips et al., in Plant Cell Tissue Organ Culture 1, 123 (1981), described the somatic embryogenesis of soybean in cell suspension or on agar. They utilized hypocotyl or epicotyl tissue for callus initiation on L2 medium. Cell suspension cultures were initiated from callus tissue in S2 medium. The cell suspension culture could be used to produce globular and heart-shaped embryos or additional callus which could form shoot buds. The formation of somatic embryos or shoot buds was reproducible using basal S2 or L2 media supplemented with 100 mg/l casein hydrolysate, 2.25 .mu.M 2,4-dichlorophenoxyacetic acid (2,4-D), 0.1 .mu.M abscisic acid (ABA), 0.1 .mu.M 2-isopropyl-4-dimethylamino5-methylphenyl-1-piperidine carboxymethyl chloride (AMO 1618) and either 15 .mu.M adenine or 0.46 .mu.M kinetin. Although somatic embryos or shoot buds were formed, on plants were obtained for any varieties of Glycine max.
Wildholm et al., in Plant Cell Reports 2, 19 (1983), describe the formation of shoots from Glycine canescens callus obtained from hypocotyls or cotyledons. Root formation did not occur, so no plantlets were obtained. The method did not produce shoots when Glycine max (soybean) was the source of the tissue. The formation of shoots from Glycine canescens tissue culture was achieved through callus induction on B5 basal medium containing 0.5 mg/l .alpha.-naphthalene acetic acid (NAA) followed in series by MS basal medium containing 0.5 mg/l of indoleacetic acid (IAA) and 5 mg/l benzyladenine (BA), and finally MS basal medium containing 0.5 mg/l BA.
Gamborg et al., in Plant Cell Reports 2, 209 (1983a), disclose somatic embryogenesis from cell suspension culture in several Glycine species including three cultivars (out of seven tested) of Glycine max. The embryoid induction medium utilized consisted of the major salts of SL, the micronutrients and vitamins of B5, 10 mg/l casamino acids, 15 .mu.M adenine sulfate, 0.2 .mu.M picloram and 0.025-0.25 .mu.M AMO 1618. It was discovered that picloram was necessary for embryo induction and that it could be replaced by 0.5 to 2.0 .mu.M 2,4-D. No embryoids were induced when the auxins NAA, 1AA or indole-3-butyric acid (IBA) were utilized in place of the auxins picloram or 2,4-D. After embryoids were induced, they were transferred to embryo growth medium which consisted of SL medium containing various combinations of cytokinins (zeatin or BA), auxins (picloram) and gibberellic acid (GA.sub.3). Embryoids which were formed went to a heart-shaped structure, but failed to develop beyond this stage on the induction medium. Transfer to the growth medium did result in the formation of roots, but shoots were not formed. The use of MS medium or addition of abscisic acid, coconut milk or change in osmolarity did not result in further development.
Gamborg et al., in Plant Cell Reports 2, 213 (1983b), describe the preparation of protoplasts from cell cultures of Glycine tabacina and Glycine soja and from leaf tissue from the soybean Glycine max cultivar (cv.) Williams 82. The protoplasts formed cells which could be induced to form heart-shaped embryos by the procedure of Gamborg et al (1983). As in the latter reference, no plantlets were formed from the procedure.
Christianson et al, in Science 222, 632 (1983a), disclose the regeneration of a plantlet from cell suspension culture of the soybean Glycine max (L.) Merrill cv. Mitchell. This appears to have been the result of a random even and appears to have resulted from a piece of clonal tissue. Immature embryos were aseptically removed from 2.5-3.0 cm pods, and embryo axes were cut into 1-2 mm pieces. These pieces were placed on a solid medium to induce callus formation. This medium consisted of MS salts, 0.5 mg/l nicotinic acid, 0.5 mg/l pyridozine, 100 mg/l thiamine, 100 mg/l inositol, 2% sucrose and 5 mg/l 2,4-D. The hard, non-friable tissue was selected for transfer to new medium and resulted in a tissue line that gave rise to hard, green, glossy, abnormal embryos. When the callus tissue was transferred from the induction medium to a N-amended medium and then transferred back to the induction medium, one exceptional piece of tissue was obtained which was covered with small embryoids. The N-amended medium consisted of the induction medium in which the two nitrogen salts of the MS salts were replaced with 20 mM ammonium citrate. Transfer of the embryoids to a medium containing 0.005 mg/l IBA and 0.2 mg/l BA gave rise to shoot formation. Transfer of the shoots to a basal medium containing 0.1 mg/l of IAA resulted in root formation to produce plantlets. This procedure does not appear to be generally applicable for regenerating soybean cultivars, but instead appears to have been a random event which may not be reproducible. Support for this analysis lies in Christianson et al.'s source of the embryogenic tissue. Christianson et al. state that "one exceptional piece of tissue" with embryoids was obtained. Since only one was obtained out of many initiated, it implies that this regeneration was a random even and could have been clonal in nature.
Copending application Ser. No. 356,222 discloses a method for regenerating several cultivars of soybean (Glycine max). In the process described in that application, an immature embryo is cultured in the light on a first medium which contains either 2,4-D, a mixture of 2,4-D and IAA, or a mixture of 2,4-D, IAA and ABA as the hormone to induce callus formation and embryoid formation from the callus. The embryoids are matured by culturing them on a second medium which is selected from a group of five media. The mature embryoids are then cultured on a third medium to induce shoot formation. The hormone utilized in the third medium may be a mixture of IBA and BA, a mixture of IAA and adenine sulfate, a mixture of IBA, BA and GA.sub.3, or a mixture of IAA, BA and GA.sub.3. Shoots are transferred to a fourth medium which is either hormone-free or contains the hormone IAA or IBA to induce the formation of roots.
Ranch, J. P., et al., In Vitro Cell Develop. Boil. 21, 653 (1985) discloses a method for regenerating several cultivars of soybean. In this process, callus tissue is initiated and somatic embryos formed by culturing immature embryos in the light (50 lux) on a first medium which contains 2,4-D. The somatic embryos are matured in the dark by culturing on a medium which contains IBA or a mixture of IBA and ABA as the hormone. The maturation medium is either liquid or solid. The embryos are germinated by culturing in the light (100 lux) on a medium containing a mixture of IBA and GA.sub.3 as the hormone. The young plants are transferred to medium containing IBA for root development and cultured in the light (1500 lux).
Soybean plants and seeds are produced by this process. The resulting soybean plants may differ from the starting plant material as a result of somoclonal variation. The pathway is also useful in that it will enable the use of various selection processes to provide further variation. Plants which are produced can be used in conventional breeding programs.